The present invention relates to a truncated mutant of the 92 kDa gelatinase which is catalytically active comparable to the full protein but, unlike the full protein, is essentially inactive against insoluble elastin.
The truncated mutant of the invention is useful for treatment of disorders requiring the removal of excess connective tissue, e.g., keloids, post-operative fibrosis, intervertebral disc injections, hypertrophic scars, wound debridement, post-surgical adhesions and various fibrotic diseases (scleroderma, idiopathic pulmonary fibrosis) and the like treatments.
(Note: Literature references on the following background information and on conventional test methods and laboratory procedures well known to the ordinary person skilled in the art, and other such state-of-the-art techniques as used herein are indicated in parentheses, and appended at the end of the specification.)
Elastin is an extracellular matrix protein composed of hydrophobic tropoelastin monomers that are highly crosslinked. Elastin provides resilience to elastic fibers. The hydrophobicity of tropoelastin monomers, as well as their extensive crosslinking, result in an insoluble elastic fiber which is highly resistant to proteolysis (1). In the normal physiologic state, elastin undergoes minimal turnover (2). However, pathologic situations exist including pulmonary emphysema (3) and abdominal aortic aneurysm (4) which are characterized by proteolytic destruction of elastic fibers.
The matrix metalloproteinases comprise a gene family of enzymes that collectively are capable of degrading all components of extracellular matrix in physiologic and pathologic states (5). These enzymes are organized into homologous domain structures, with some differences in number of domains. All members share a zymogen domain and a zinc-binding catalytic domain. As presently recognized, this family consists of fibroblast, neutrophil, and a breast carcinoma-derived (6) collagenase, a 92 kDa gelatinase (also called gelatinase B and MMP-9), a 72 kDa gelatinase (also called gelatinase A and MMP-2), three stromelysins, macrophage metalloelastase, matrilysin (also known as PUMP and MMP-7), and a recently described 66 kDa membrane-type metallproteinase (7). Four members of this gene family have the capacity to degrade insoluble elastin. These are the 92 kDa gelatinase (8,9), the 72 kDa gelatinase (8,9), matrilysin (9), and macrophage metalloelastase (10,11).
The issue of substrate specificity has received considerable attention recently in matrix metalloproteinase biology. The structural determinants within these enzymes which confer the ability to degrade various substrates appear to be localized within discrete domains. For example, the ability of the collagenases to degrade triple-helical collagen requires the presence of the C-terminal hemopexin-like domain (12-14). In contrast, the stromelysins degrade a variety of substrates in a manner which is independent of the C-terminal hemopexin-like domain (13, 15-17). Unique to the 72 kDa and 92 kDa gelatinases is an additional domain composed of three fibronectin type II repeats inserted in tandem within the zinc-binding catalytic domain. This fibronectin-like domain is required for the gelatinases to bind efficiently to type I gelatin and type IV collagen (18-21). Matrilysin is the simplest member of this family of enzymes in that it contains only a zymogen domain and a catalytic zinc-binding domain.
A 92 kDa type IV collagenase (gelatinase) and the cDNA clone representing the full-length protein is disclosed in U.S. Pat. No. 4,992,537.